Multiple color emissions from a single bioluminescence system have often been the focus for extensive studies either for elucidation of luciferase or fluorescent protein structure-function relationships. Additionally, they have been studied for the development of improved sensors.
Bioluminescence derived from luciferase-catalyzed reactions and fluorescent protein emissions has been widely used in life sciences for both basic and applied studies. For most luciferases, the required luciferins that undergo luciferase-catalyzed oxidation are unusual metabolites specialized for bioluminescence reactions. If used for signaling or imaging purposes, it may be difficult or inconvenient to provide these unusual luciferins to the systems under investigation, especially for in vivo studies.
In this way, bacterial luciferase-catalyzed bioluminescence has some unique advantages over other luciferase systems. Oxygen and reduced riboflavin 5′-phosphate (FMNH2), e.g., two of the three substrates required by bacterial luciferase for producing luminescence, are readily available in various cell types. The third substrate for the bacterial luciferase-catalyzed oxidative process, long-chain aliphatic aldehyde, cannot be generated by most other cell types. However, the long-chain aldehydes may be synthesized by the luxCDE genes in luminous bacteria. Further, these long-chain aldehydes have very high binding affinities for luciferase, with Km below micromolar levels, and due to their highly hydrophobic nature, can easily diffuse through cell membranes for in vivo imaging applications.
The aldehyde-generating genes may be cloned along with luxAB genes for bacterial luciferase α and β subunits into the cell systems. Moreover, the codon-optimized Vibrio harveyi luciferase lux genes and the frp gene for flavin reductase P (FRP), which generates FMNH2 from FMN using NADPH as a reductant, have been successfully expressed in mammalian HEK 293 cells with significant signals for bioluminescence imaging. The lux genes have been well characterized, and the structures and reaction mechanisms of bacterial luciferase have also been extensively studied, making bacterial luciferase particularly suitable for serving as a reporting system.
However, as applied for current in vivo systems, the normal 490 nm emission by bacterial luciferase suffers from two disadvantages, namely absorption by some cellular components and a lower ability to penetrate tissue.
In this connection, changes in the color of bacterial luciferase bioluminescence have previously been reported in three lines of studies. First, replacements of the FMNH2 substrate by reduced 2-thioFMN and iso-FMN were found to shift the 490 nm peak of the bioluminescence to 530 and 470 nm, respectively. Second, certain mutations of the bacterial luciferase active center structure also led to changes of the bioluminescence color. In another study, the luciferase α subunit was randomly mutated and five luciferase variants were found to emit bioluminescence with significant but limited shifts from that of the normal 490 nm emission. This finding suggested that the excited emitter is bound to luciferase and its emission is sensitive to the environment of the luciferase active site. The luciferase α subunit was also subjected to controlled single- to triple-residue mutations for altering the emission spectrum. Third, bacterial bioluminescence color, in vitro and in vivo, may also be changed by the formation of a non-covalent complex of luciferase with specific fluorescent proteins co-existing in luminous bacteria. These fluorescent proteins include a lumazine protein from Photobacterium phosphoreum and Photobactreum leiognothi, two forms of yellow-fluorescent proteins, and a blue-fluorescent protein from Vibrio fischeri strain Y1.
Some have used certain flavin analogs for altering the bacterial luciferase emission color in vitro, however, it is not applicable to in vivo systems. Additionally, due to the rather stringent specificity of luciferase for the flavin substrate, color changes of bacterial luciferase bioluminescence by flavin analogs other than 2-thioFMN and iso-FMN have not been reported. Others have changed the luciferase active site structure as a way to alter bacterial luminescence color by either random mutation or site-directed mutagenesis, and producing luciferase variants that generally have substantially reduced activities and their emission spectral changes are limited (up to 12 nm shifts to the red).
Others have blue-shifted, by about 10- and 20-nm peak, the normal 490-nm bioluminescence of luciferase by the blue fluorescent protein and lumazine protein, respectively, and red shifted to near 540 nm by the yellow fluorescent proteins. Lumazine protein and yellow fluorescent protein each interacts specifically and directly with luciferase reaction intermediate(s) to alter not only the color but also the reaction kinetics of the luciferase-catalyzed emission. This may be best demonstrated by the single turnover reaction by luciferase in the presence of yellow fluorescent protein, in which the decay rates of the 490 nm normal bioluminescence by luciferase and the 540 nm emission from yellow fluorescent protein were distinct. Therefore, the emission spectrum actually changes over time and resonance energy transfer alone cannot account for the change of emission color.
Prior studies have reported that lumazine protein, blue and yellow fluorescent proteins do not have the ability to alter the bioluminescence spectra of all known bacterial luciferases; one species of fluorescent protein can change the bioluminescence color of only one or a very limited few species of luciferase. Moreover, it has been reported that their non-covalent interactions with corresponding luciferases are sensitive to perturbations by factors such as temperature and concentration.
Although various compositions and methods for altering the color of luciferase luminescence are known to the art, all, or almost all of them suffer from one or more than one disadvantage and have shortcomings. Therefore a need has arisen for compositions and methods for changing the color of luciferase bioluminescence which corrects the problems identified above, and enables its use in vivo or as part of multi-color sensor systems.